Development of Quantitative Raman Imaging Techniques to Study
 Intracellular Pharmacokinetics of Anticancer Agents, 10-9219

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Principal Investigators
Jian Ling
Michael A. Miller

Inclusive Dates: 10/01/00 - 09/30/01

Background - The determination of drug mechanisms at the cellular level is a problem of great importance to the evaluation and development of new anticancer agents. Recently, the implementation of rational drug designs, combinatorial chemistry techniques, and high throughput screening have led to large numbers of new candidate drugs. Currently, limited cost-effective and efficient approaches restrict understanding the details of how these candidate drugs work at the cellular level. This lack of good approaches requires pharmaceutical companies to spend millions of dollars in animal and clinical studies to assess a candidate drug.

Approach - The objective of this project is to develop direct Raman imaging as a cost-effective tool to study drug mechanisms at the cellular level. The application of direct Raman imaging was studied to determine drug distribution in single living cells. To extract useful information from a recorded Raman image, a model was developed to describe the degradation of Raman signals during imaging: the nonuniform illumination of the laser excitation source, blurring by the microscope system, the influence of the fluorescence background, and the disturbance of additive signal-dependent Gaussian noise. Using this model, special-purpose image-processing algorithms were developed to restore the Raman images. Taxol, an important anticancer agent whose mechanisms at the cellular level have been well studied, was used to evaluate the capabilities of direct Raman imaging. Raman images were obtained from a MDA-435 cancer cell before, during, and after the drug treatment.

Accomplishments - The Raman images show how the distribution of Taxol in a living tumor cell changes with time. It was also found that Taxol does not enter the cell nucleus but concentrates around the cell centrosome and near the cell membrane (refer to the illustrations below). This finding is well explained by the binding characteristics of the Taxol and its molecular target, the microtubules. This result demonstrated the feasibility of using direct Raman imaging to determine the intracellular distribution of a drug. From the drug distribution, its mechanism and intracellular pharmacokinetics can be studied.

 

 
White light image of a breast tumor cell showing the cell structure. (Middle)  Raman image of the same cell, recorded 1.75 hours after exposure to Taxol, shows the distribution of Taxol. The highest intensity of the Raman signal is shown in red, while deep blue indicates the lowest intensity. The superimposition of the images in preceding illustrations shows the Taxol distribution in the cell. 

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